Glutathione in Cerebral Microvascular Endothelial Biology and Pathobiology: Implications for Brain Homeostasis

The integrity of the vascular endothelium of the blood-brain barrier (BBB) is central to cerebrovascular homeostasis. Given the function of the BBB as a physical and metabolic barrier that buffers the systemic environment, oxidative damage to the endothelial monolayer will have significant deleterious impact on the metabolic, immunological, and neurological functions of the brain. Glutathione (GSH) is a ubiquitous major thiol within mammalian cells that plays important roles in antioxidant defense, oxidation-reduction reactions in metabolic pathways, and redox signaling. The existence of distinct GSH pools within the subcellular organelles supports an elegant mode for independent redox regulation of metabolic processes, including those that control cell fate. GSH-dependent homeostatic control of neurovascular function is relatively unexplored. Significantly, GSH regulation of two aspects of endothelial function is paramount to barrier preservation, namely, GSH protection against oxidative endothelial cell injury and GSH control of postdamage cell proliferation in endothelial repair and/or wound healing. This paper highlights our current insights and hypotheses into the role of GSH in cerebral microvascular biology and pathobiology with special focus on endothelial GSH and vascular integrity, oxidative disruption of endothelial barrier function, GSH regulation of endothelial cell proliferation, and the pathological implications of GSH disruption in oxidative stress-associated neurovascular disorders, such as diabetes and stroke

Disruption of Pyridine Nucleotide Redox Status During Oxidative Challenge at Normal and Low-Glucose States: Implications for Cellular Adenosine Triphosphate, Mitochondrial Respiratory Activity, and Reducing Capacity in Colon Epithelial Cells

We recently demonstrated that menadione (MQ), a redox cycling quinone, mediated the loss of mitochondrial glutathione/glutathione disulfide redox balance. In this study, we showed that MQ significantly disrupted cellular pyridine nucleotide (NAD+/NADH, NADP+/NADPH) redox balance that compromised cellular ATP, mitochondrial respiratory activity, and NADPH-dependent reducing capacity in colonic epithelial cells, a scenario that was exaggerated by low glucose. In the cytosol, MQ induced NAD+ loss concurrent with increased NADP+ and NAD kinase activity, but decreased NADPH. In the mitochondria, NADH loss occurred in conjunction with increased nicotinamide nucleotide transhydrogenase activity and NADP+, and decreased NADPH. These results are consistent with cytosolic NAD+-to-NADP+ and mitochondrial NADH-to-NADPH shifts, but compromised NADPH availability. Thus, despite the sacrifice of NAD+/NADH in favor of NADPH generation, steady-state NADPH levels were not maintained during MQ challenge. Impairments of cellular bioenergetics were evidenced by ATP losses and increased mitochondrial O2 dependence of pyridine nucleotide oxidation–reduction; half-maximal oxidation (P50) was 10-fold higher in low glucose, which was lowered by glutamate or succinate supplementation. This exaggerated O2 dependence is consistent with increased O2 diversion to nonmitochondrial O2 consumption by MQ-semiquinone redox cycling secondary to decreased NADPH-dependent MQ detoxication at low glucose, a situation that was corrected by glucose-sparing mitochondrial substrates. Antioxid. Redox Signal. 14, 2151–2162

A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs

Bafilomycin A1, a specific inhibitor of vacuolar-type proton pump, induced perinuclear distribution of lysosomes.

<p><b>(A)</b> DU145 cells were treated for 16 hours with DMSO, 25 μM EIPA, 0.1 μM bafilomycin, 1 μM niclosamide or 50 μM chloroquine. Then, pH was dropped to 6.4 in all conditions for an additional 2 hours. Cells were then fixed and immunostained for LAMP1 (red), DAPI (blue) and actin (green). <u>Scale bars: 10</u> μ<u>m</u>. <b>(B)</b> DU145 cells were treated for 16 hours with DMSO, bafilomycin A1 (Baf), niclosamide (Nic), or EIPA in serum free (SF) and complete media (S). Lysosome distribution was calculated “mean ring spot count channel 3” using the Cellomics Imager. Error bars represent the SD from at least 3 independent experiments. * denotes statistical significance (p<0.05) relative to treatment with DMSO.</p

Incubation with niclosamide prevents acidic pHe and growth factor-induced lysosome trafficking.

<p><b>(A)</b> DU145 cells were treated with pHe 6.4, 33 ng/mL HGF or 100 ng/mL EGF in the presence or absence of 0.5 μM niclosamide. Cells were fixed and stained for DAPI (blue), actin (green), and LAMP-1 (red). In control and in EGF or HGF-treated cells the lysosomes are located at the periphery (white arrows) whereas in niclosamide-treated cells the lysosomes are around the nucleus (white arrowheads). Scale bars: <u>10</u> μ<u>m</u>. DU145 cells were treated overnight with varying concentrations of niclosamide diluted in <b>(B)</b> low pH media (pH 6.4), <b>(C)</b> media containing 33 ng/mL HGF, or <b>(D)</b> media containing 100 ng/mL EGF and relative lysosome distribution was calculated using the cellomics imager. Quantification of lysosome position is shown as the average of the calculated “mean ring spot count channel 3”. Error bars represent the SD from at least 3 independent experiments. * denotes statistical significance (p<0.01) versus niclosamide. <b>(E)</b> DU145 cells were treated with 1 μM Niclosamide over time and relative lysosome distribution was calculated using the cellomics imager. Quantification of lysosome position is shown as the average of the calculated “mean ring spot count channel 3”. Error bars represent the SD from at least 3 independent experiments.</p

Niclosamide inhibits lysosome trafficking, motility, and invasion in glioma A172 cells.

<p><b>(A)</b> A172 cells were treated for 8 hours with DMSO, 25 μM EIPA, or varying concentrations of niclosamide diluted in low pH media (pH 6.4). Next, cells were fixed and stained for LAMP-1 (red), actin (green) and DAPI (blue). Lysosomes in DMSO control have a peripheral location whereas in niclosamide or EIPA-treated cells they are located around the nucleus (as indicated by arrows). <u>Scale bars: 10</u> μ<u>m</u>. <b>(B)</b> A172 cells were plated in collagen-coated 96 well plates and allowed to form a confluent monolayer prior to wounding. Next, 20% matrigel was added to the wells for which invasion is to be studied. Cells were allowed to migrate or invade in the presence of DMSO, 33 ng/mL HGF or 100 ng/mL EGF in the presence or absence of 0.3 μM niclosamide. Motility and invasion were calculated by IncuCyte Imager and the relative wound density percentage at 24 hours post wound is shown. Error bars represent the SD from at least 3 independent experiments. * p<0.05 treatment versus serum free or DMSO, ** p<0.01 treatment versus serum free or DMSO.</p

Niclosamide inhibits the acidic pHe-induced cathepsin B secretion and prevents DQ collagen IV cleavage.

<p><b>(A)</b> DU145 cells were treated for 24 hours with DMSO in pH 7.4 media, DMSO in pH 6.4 media or 1 μM niclosamide in pH 6.4 media. The activity of secreted cathepsin B was determined. * denotes statistical significance (p<0.05) versus DMSO/pH 7.4. Error bars represent the SD from at least 3 independent experiments. <b>(B)</b> Cells were treated with DMSO in pH 7.4 media, DMSO in pH 6.4 media or 1 μM niclosamide in pH 6.4 media. Whole cell lysates were collected and Western blot analysis was performed for the indicated proteins. <b>(C)</b> DU145 cells were grown for two days on matrigel containing DQ collagen IV and then incubated for an additional 24 hours with 33 ng/mL HGF or 0.3 μM niclosamide. Cells were fixed and stained for actin (red). Green represents cleaved DQ-collagen IV. Representative confocal images are shown.</p